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RESEARCH ARTICLE
Active transforming growth factor-β2 in the
aqueous humor of posterior polymorphous
corneal dystrophy patients
Andrea Stadnikova1, Lubica Dudakova1, Pavlina Skalicka2, Zdenek Valenta3,
Martin Filipec4, Katerina Jirsova1*
1 Laboratory of the Biology and Pathology of the Eye, Institute of Inherited Metabolic Disorders, First Faculty
of Medicine, Charles University and General University Hospital in Prague, Czech Republic, 2 Department of
Ophthalmology, First Faculty of Medicine, Charles University and General University Hospital in Prague,
Czech Republic, 3 Department of Medical Informatics & Biostatistics, Institute of Computer Science, Czech
Academy of Sciences, Prague, Czech Republic, 4 European Eye Clinic Lexum, Prague, Czech Republic
* [email protected]
Abstract
Purpose
Posterior polymorphous corneal dystrophy (PPCD) is characterized by abnormal prolifera-
tion of corneal endothelial cells. It was shown that TGF-β2 present in aqueous humor (AH)
could help maintaining the corneal endothelium in a G1-phase-arrest state. We wanted to
determine whether the levels of this protein are changed in AH of PPCD patients.
Methods
We determined the concentrations of active TGF-β2 in the AH of 29 PPCD patients (42 sam-
ples) and 40 cadaver controls (44 samples) by ELISA. For data analysis the PPCD patients
were divided based on either the molecular genetic cause of their disease as PPCD1 (37 sam-
ples), PPCD3 (1 sample) and PPCDx (not linked to a known PPCD loci, 4 samples) or on the
presence (17 samples) or absence (25 samples) of secondary glaucoma or on whether they
had undergone penetrating keratoplasty (PK, 32 samples) or repeated PK (rePK, 7 samples).
Results
The level of active TGF-β2 in the AH of all PPCD patients (mean ±SD; 386.98 ± 114.88 pg/ml)
in comparison to the control group (260.95 ± 112.43 pg/ml) was significantly higher (P = 0.0001).
Compared to the control group, a significantly higher level of active TGF-β2 was found in the
PPCD1 (P = 0.0005) and PPCDx (P = 0.0022) groups. Among patients the levels of active TGF-
β2 were not significantly affected by gender, age, secondary glaucoma or by the progression of
dystrophy when one or repeated PK were performed.
Conclusion
The levels of active TGF-β2 in the AH of PPCD patients are significantly higher than control
values, and thus the increased levels of TGF-β2 could be a consequence of the PPCD phe-
notype and can be considered as another feature characterizing this disease.
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OPENACCESS
Citation: Stadnikova A, Dudakova L, Skalicka P,
Valenta Z, Filipec M, Jirsova K (2017) Active
transforming growth factor-β2 in the aqueous
humor of posterior polymorphous corneal
dystrophy patients. PLoS ONE 12(4): e0175509.
https://doi.org/10.1371/journal.pone.0175509
Editor: James Fielding Hejtmancik, National Eye
Institute, UNITED STATES
Received: September 14, 2016
Accepted: March 27, 2017
Published: April 17, 2017
Copyright: © 2017 Stadnikova et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
file.
Funding: The research leading to these results has
received funding from the Norwegian Financial
Mechanism 2009-2014 and the Ministry of
Education, Youth and Sports under Project
Contract no. MSMT-28477/2014, project 7F14156
(AS, KJ). This study was further supported by the
PROGRES-Q26/LF1 program of the Charles
University (KJ, LD). ZV was supported by the long-
Page 2
Introduction
Posterior polymorphous corneal dystrophy (PPCD) is a bilateral disorder affecting all layers of
the cornea but most severely its posterior part, i.e., the endothelium, Descemet’s membrane
and the deepest stromal layers [1, 2]. PPCD is genetically heterogeneous: PPCD1 (OMIM
#122000) is caused by mutations in the OVOL2 promoter [3], PPCD2 (OMIM #609140) has
been associated with mutations in COL8A2 [4] and PPCD3 (OMIM #609141) with mutations
in ZEB1 genes [5].
PPCD affects at least 1:100,000 inhabitants in the Czech Republic and most patients carry a
disease-causing founder mutation in OVOL2 [3]. Several PPCD3 families have also been iden-
tified [6–10]. In one family linkage exclusion to the PPCD1 locus and a lack of mutations in
the coding sequence of ZEB1 and COL8A2 suggest the possibility of the existence of a novel
disease locus [10, 11].
The human corneal endothelium is a monolayer of flat hexagonal cells, which are normally
arrested in the G1-phase of the cell cycle but retain their proliferative capacity [12] that may be
renewed in vivo and in vitro by the disruption of cell-cell contacts and by the addition of
growth factors into the anterior chamber or culture medium [13–15].
The corneal endothelial cells of PPCD patients lose their original characteristics and acquire
an epithelial- or fibroblast-like phenotype [1, 16, 17]. Proliferating abnormal cells extend out-
wards from the cornea over the trabecular meshwork; often leading to secondary glaucoma [1,
18]. The epithelial features of these aberrant cells include abundant desmosome formation,
microvilli on their apical surface and the expression of keratins [17, 19]. Descemet’s membrane
becomes irregularly thickened with the presence of a posterior collagenous layer and altered
collagen expression [2, 20]. It has been confirmed that the recurrence of PPCD in patients
after penetrating keratoplasty (PK) is caused by the overgrowth of pathological host endothe-
lium [21]. The precise molecular mechanisms behind the epithelialization of the corneal endo-
thelium occurring in PPCD still remain unclear.
Transforming growth factor-beta (TGF-β) signaling is involved in almost all physiological
and pathological cell behavior including regulation of immunity, differentiation, proliferation,
migration, and production of the extracellular matrix [22–24]. It was shown that TGF-β2, the
major isoform of the TGF-β family, is secreted into the aqueous humor (AH) as an inactive
precursor by the trabecular meshwork and ciliary body [25, 26]. About 2% of the total TGF-β2
is then available in AH in its activated form [27].
Generally, activators of latent TGF-β2 include thrombospondin, matrix metalloproteinases,
integrins, reactive oxygen species and/or an acidic environment [28]. Corneal endothelial cells
express all three TGF-β receptors [29]. Controversy exists as to whether adult corneal endothe-
lial cells produce TGF-β2 [30–32]. TGF-β2 has the ability to suppress S-phase entry in cultured
corneal endothelial cells by blocking the degradation of p27kip1 cyclin-dependent kinase
inhibitor and thus may contribute to G1-phase arrest in these cells in vivo [12, 33, 34]. In ocu-
lar disorders, the level of active TGF-β2 in human AH has been found to be both higher (pri-
mary open-angle glaucoma, keratoconus) [35, 36] and lower (uveitis, endothelial immune
reactions following PK) [37, 38] than in controls.
The aim of this work was to evaluate the levels of active TGF-β2 in the AH of PPCD
patients. We were also interested to determine whether the level of TGF-β2 depends on the
progression of PPCD, characterized by the necessity to perform PK and/or by the concurrent
presence of glaucoma.
The level of active TGF-β2 in AH of PPCD patients is higher compared to controls
PLOS ONE | https://doi.org/10.1371/journal.pone.0175509 April 17, 2017 2 / 11
term strategic development financing of the
Institute of Computer Science (RVO: 67985807).
LD was supported by UNCE 204011/2012 and
GACR 17-12355S.
Competing interests: The authors have declared
that no competing interests exist.
Page 3
Material and methods
Patients and specimen preparation
The study was approved by the Ethics Committee of the General University Hospital in Prague
and Charles University (8/02 GACR, 1838/08 S-IV), and followed the tenets set out in the Dec-
laration of Helsinki. Based on Czech legislation on specific health services (the law Act No.
372/2011 Coll.), the informed consent is not required if presented data are anonymized in the
form, nevertheless, written informed consents were obtained from all subjects collected from
2008. None of the body donors were from a vulnerable population and all donors or next of
kin provided written informed consent that was freely given.
The clinical diagnosis of PPCD was based on the presence of typical signs observed at slit
lamp examination such as the presence of vesicular and geographical lesions associated with
irregularities of the otherwise smooth posterior corneal surface and opacities at the level of
Descemet’s membrane [1, 39]. The diagnosis of glaucoma was based on the results of routine
clinical examination.
AH samples from PPCD patients were collected during surgery (PK, cataract surgery or
glaucoma surgery) at the Department of Ophthalmology, First Faculty of Medicine and Charles
University and General University Hospital in Prague between the years 1994 and 2008 and
stored at -80˚C until analysis. All samples were frozen within 3 hours after their collection. In
total, 42 AH samples were obtained from 29 patients of Caucasian Czech origin (17 females, 12
males). Twenty two patients provided samples from one eye and seven patients from both eyes.
In 4 out of the 22 patients which provided samples from one eye AH was collected repeatedly
during multiple surgeries on the same eye (2 patients provided 3 samples from one eye and 2
patients provided 2 samples from one eye, i.e., in total 10 samples). The mean age ± SD at the
time of surgery in the group of all PPCD patients (PPCD_all) was (46.61 ± 20.04 years).
On the basis of our previously reported molecular genetic investigations [3, 7], the patients
were divided into three groups: PPCD1 (37 samples), PPCD3 (1 sample) and PPCDx (individ-
uals from a family with an unknown molecular genetic cause not linked to any of the known
PPCD loci, 4 samples). The PPCD_all patients were further grouped according to the presence
or absence of secondary glaucoma, PPCD_glaucoma+ (17 samples) and PPCD_glaucoma- (25
samples), respectively, and whether or not they underwent corneal transplantation previously
or on the day of AH sample collection, PPCD_PK+ (32 samples) and PPCD_PK- (10 samples),
respectively. The PPCD_PK- group underwent either cataract or glaucoma surgery. Finally,
patients that underwent repeated PK were grouped into PPCD_rePK (7 samples).
Control AH samples obtained from cadaveric donors were used for subsequent measure-
ments of TGF-β2 levels [40]. In total, 44 samples were obtained from 40 donors (13 females and
27 males; mean age ± SD; 60.48 ± 12.79 years) with no history of corneal disease during the prep-
aration of donor corneas in the Ocular Tissue Bank, General University Hospital in Prague. The
samples were collected as follows: 18 samples from one eye, 18 samples pooled from the right
and left eyes and 8 samples from both eyes separately. Control samples were collected between
the years 2003 and 2009 within 24 hours after donor death, as performed in our previous studies
on PPCD [2, 17]. The collection followed the regulations established for tissues collected for use
in corneal transplantation, AH samples were not collected specifically for this study. All samples
were frozen within 2 hours after their collection and stored at -80˚C until analysis.
TGF-β2 immunoassay
The concentration of active TGF-β2 (without activation of the latent form) was determined
using a quantitative sandwich ELISA kit according to the manufacturer‘s instructions (SB250,
The level of active TGF-β2 in AH of PPCD patients is higher compared to controls
PLOS ONE | https://doi.org/10.1371/journal.pone.0175509 April 17, 2017 3 / 11
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Quantikine1, R&D Systems, UK). Standards and samples were tested in 50 μl volume. Mea-
surements were performed 2–4 times for each sample (based on the obtained volume of AH).
The optical density was measured using an Infinite M200 spectrophotometer (Tecan, Manne-
dorf, Switzerland).
Statistical analysis
Descriptive statistics are reported as N, mean, standard deviation and 95% confidence interval.
The normality of the distribution of the TGF-β2 values was assessed using both the Shapiro-
Wilk and the Lomnicki-Jarque-Bera normality tests. P values of< 0.05 were considered to be
statistically significant. Differences in active TGF-β2 levels were assessed using Linear Mixed-
Effects Models for analyzing data involving repeated measurements on the same individuals,
as implemented in the ’nlme’ package in R [41]. Random effects were associated with subjects
contributing repeated measurements over time. In order to avoid bias in estimating variance
components, Restricted Maximum Likelihood (REML) was used in modeling. Analysis of Var-
iance (ANOVA) and Wald-type tests were used to assess the significance of the model parame-
ters. The results are expressed as differences (d) in the mean active TGF-β2 values between
individual groups. No valid data or data groups were arbitrarily excluded from the analysis.
Low frequencies in the observed data groups (1 sample in the PPCD3 group) resulting from
random sampling were left for statistical testing to provide evidence for inference. Confound-
ing effect of age on the levels of TGF-β2 within the control group was assessed using linear
regression modeling involving independent data where multiple observations per patient were
replaced with the corresponding average value, and also using linear mixed model regression
analysis involving the original data with possibly repeated observations per patient. Welch
two-sample t-test involving independent data was used to analyze differences in age distribu-
tion between the groups of control and PPCD_all patients.
Results
The mean levels of active TGF-β2 (mean ± SD) in the AH of the control group and all PPCD
patients were 260.95 ± 112.43 pg/ml and 386.98 ± 114.88 pg/ml, respectively. All descriptive
characteristics of the active TGF-β2 levels within the various target groups are presented in
Table 1. The distributions of active TGF-β2 levels within the patient groups and controls are
shown in Fig 1. None of the considered confounding factors, namely the age, gender, history
of PK, repeated PK and glaucoma, proved to exert a statistically significant effect on the mean
levels of TGF-β2. The best data summary was therefore obtained from unadjusted models that
did not involve the confounding factors.
Considering all PPCD patients as one group, analysis of variance revealed significant differ-
ences in the mean levels of active TGF-β2 in the AH between the PPCD patients and controls
(P = 0.0001). The estimated mean difference (d) in TGF-β2 values between these two groups
was 120.4 pg/ml, P = 0.0001. Considering the three groups (i.e., PPCD1, PPCD3 and PPCDx)
against the baseline (controls), analysis of variance again detected statistically significant differ-
ences in the mean levels of TGF-β2 (P = 0.0003). More specifically, the TGF-β2 levels in the
AH were shown to be significantly higher in PPCD1 patients (d = 106.0 pg/ml, P = 0.0005)
and in PPCDx patients (d = 216.6 pg/ml, P = 0.0022), as compared to the controls. The esti-
mated mean difference in TGF-β2 values between the PPCD3 value and the controls was
d = 199.2 pg/ml, P = 0.1040. The estimated differences in the mean TGF-β2 levels along with
95% confidence limits are shown in Table 2.
Analysis of variance based on the fully adjusted model demonstrated that the presence of
PPCD was the only factor that influenced the levels of active TGF-β2 statistically significantly
The level of active TGF-β2 in AH of PPCD patients is higher compared to controls
PLOS ONE | https://doi.org/10.1371/journal.pone.0175509 April 17, 2017 4 / 11
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(P = 0.0003). The levels were not altered statistically significantly by the presence of secondary
glaucoma (P = 0.3067) or by the development of PPCD into a state where it was necessary to
undergo PK (P = 0.4228) or repeated PK (P = 0.2879). Similarly, neither gender (P = 0.2138)
nor age (P = 0.2059) proved to have influenced the levels of active TGF-β2 statistically signifi-
cantly. A summary of these results is shown in Table 3.
Obtained results show statistical significance between control and PPCD_all groups con-
cerning the age (P = 0.002; Welch Two Sample t-test), however the effect of age within the con-
trol group is statistically insignificant (P = 0.675 using a linear model for independent data
with averaged multiple responses per individual) and thus the age is not a significant con-
founder of TGF-β2 levels. Our PPCD samples were collected between the years 1994 and 2008,
and thus a portion of samples is considerably older than control samples (collected between
2003 and 2009). The mean levels of active TGF-β2 (mean ± SD) in the AH of the PPCD older
specimens (collected between 1994 and 2002) and younger specimens (collected between 2003
and 2008) were 368.12 ± 124.62 pg/ml and 407.18 ± 104.16 pg/ml, respectively. Difference in
active TGF-β2 levels between younger and older PPCD specimens is not significant
(P = 0.3669; Welch Two Sample t-test) and therefore the period of storage is not a confounding
factor.
Discussion
Although the precise molecular mechanisms leading to the PPCD phenotype are still unex-
plained, the histopathological abnormalities, particularly changes affecting the corneal endo-
thelium, are considered to be a consequence of cell dysregulation due to a disease-causing
mutation. As TGF-β2 influences cell differentiation, proliferation, migration and extracellular
matrix production [22, 23], we aimed to determine whether there are changes in the levels of
Table 1. Descriptive statistics.
Active TGF-β2 [pg/ml]
Group N* Mean SD 95% LCL† 95% UCL‡
Controls 40 260.95 112.43 224.99 296.91
PPCD_all 29 386.98 114.88 343.28 430.67
PPCD1 25 373.39 116.39 325.35 421.44
PPCD3§ 1 465.00 - - -
PPCDx 3 474.17 73.81 290.80 657.53
Controls female 13 224.46 104.89 161.08 287.84
PPCD_all female 17 378.26 86.59 333.74 422.79
Controls male 27 278.52 113.57 233.59 323.45
PPCD_all male 12 399.32 149.68 304.22 494.42
PPCD_PK+ 22 378.38 108.91 330.09 426.67
PPCD_PK- 7 414.00 137.66 286.69 541.31
PPCD_rePK+ 2 335.5 67.18 -268.04 939.04
PPCD_glaucoma+ 11 383.98 130.21 296.51 471.46
PPCD_glaucoma- 18 388.81 108.42 334.89 442.72
* Number of samples.* Multiple samples from the same patient are interpreted as an average value and presented as one sample.† Lower 95% confidence limit for the mean.‡ Upper 95% confidence limit for the mean.§ There is only one patient in the PPCD3 group; it is not possible to calculate descriptive statistics.
https://doi.org/10.1371/journal.pone.0175509.t001
The level of active TGF-β2 in AH of PPCD patients is higher compared to controls
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Fig 1. Active transforming growth factor (TGF)-β2 levels in the AH measured by ELISA. (A) Distribution of the active TGF-β2 levels in the AH
of controls and all posterior polymorphous corneal dystrophy patients (PPCD_all), and (B) of controls and the groups of patients with different
molecular genetic causes: PPCD1, PPCD3, and PPCDx. Multiple data points from the same patient were averaged and the average value
presented as one data point. Active TGF-β2 levels are presented as data distribution and box plots enhanced with mean ± SD. Displayed P-values
reflect statistical significance of observed differences in the mean values of TGF-β2 obtained as fixed effects in otherwise unadjusted linear mixed-
effects model.
https://doi.org/10.1371/journal.pone.0175509.g001
The level of active TGF-β2 in AH of PPCD patients is higher compared to controls
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active TGF-β2 in the AH of PPCD patients. We found that the mean concentration of TGF-β2
in the AH of PPCD patients is significantly higher compared to that of the control group. The
levels of TGF-β2 were not altered by gender, age, secondary glaucoma or the progression of
dystrophy to a clinical state in which PK was necessary.
Cell-cell contact inhibition and active TGF-β2 maintain the mature corneal endothelium in
a non-proliferative state (G1-phase arrest) through the activity of cyclin-dependent kinase
inhibitors [34, 42]. In PPCD, aberrant cells exhibit proliferative activity and increased migra-
tion [1, 21]. Interestingly, here we have found that in PPCD the proliferation of aberrant cells
is not associated with lower, but rather significantly higher TGF-β2 levels in AH compared to
the control group; however, in 81% of samples the levels of TGF-β2 do not exceed 500 pg/ml, a
concentration that was able to reduce the proliferation of cultured human corneal endothelial
cells [43]. The responses of corneal endothelial cells in healthy endothelium and aberrant cells
present in PPCD to TGF-β2 could differ. It is not clear whether the elevation of TGF-β2 is a
prerequisite for the cell transition (epithelialization), or, more probably, whether higher levels
of TGF-β2 may arise as a consequence of endothelial cell epithelialization. In the second case,
TGF-β2 may be produced by epithelial-like cells. The expression of TGF-β2 by epithelial cells
has been repeatedly confirmed, even in the corneal epithelium [44–46]. It is probable that once
the primary changes (epithelialization) appear, they may escalate other, already ongoing
changes characteristic of the histopathology of PPCD.
In terms of the phenotypic switch occurring in PPCD, the role of TGF-β2 should be consid-
ered due to its participation in an epithelial- or endothelial-to-mesenchymal transition and, in
reverse, in a mesenchymal-to-epithelial transition [47, 48]. The corneal endothelium originates
from the neuroectoderm with an important contribution from the mesoderm, but its pheno-
typical heterogeneity is large as corneal endothelial cells express epithelial, neuronal and meso-
thelial markers [17, 49–51]. In PPCD, aberrant cells exhibit increased migration and
Table 2. Analysis of variance based on the unadjusted model for the active TGF-β2 values in individual PPCD groups.
Disease Group Estimated difference in TGF-β2 values relative to Controls (pg/ml) 95% LCL* (pg/ml) 95% UCL† (pg/ml) P-value
PPCD1 106.04 48.10 163.98 0.0005
PPCD3 199.15 -42.04 440.33 0.1040
PPCDx 216.61 80.88 352.34 0.0022
* Lower 95% confidence limit for the mean.
† Upper 95% confidence limit for the mean.
https://doi.org/10.1371/journal.pone.0175509.t002
Table 3. Influence of different independent factors on the levels of active TGF-β2 analyzed by analysis of variance based on the fully adjusted
model.
Covariate DF* Numerator†, Denominator† F-statistic P-value
(Intercept) 1, 64 534.4917 <0.0001
PPCD 3, 64 7.2643 0.0003
Age 1, 13 1.7728 0.2059
Gender 1, 64 1.5767 0.2138
PK 1, 13 0.6850 0.4228
Repeated PK 1, 13 1.2279 0.2879
Glaucoma 1, 13 1.1323 0.3067
* Degrees of freedom.
† Numbers representing degrees of freedom for F-statistics.
https://doi.org/10.1371/journal.pone.0175509.t003
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proliferative activity and gain epithelial-like (as shown morphologically and confirmed by the
expression of keratin filaments) [1, 17] and fibroblast-like features (as shown morphologically)
[1, 16]. Due to this large phenotypical divergence in both the original and aberrant cells, it is
difficult to specify the type of transition occurring in PPCD. Recently, however, an association
between PPCD1 and mutations in the promoter of OVOL2 has been demonstrated. Mutations
apparently lead to aberrant OVOL2 expression during endothelial cell development [3]. Tran-
scription factor OVOL2 is one of the key inducers of the mesenchymal-to-epithelial transition
[52].
Numerous glaucoma studies have shown that the levels of both total and active TGF-β2 in
the AH of patients with open-angle glaucoma are elevated in comparison to control specimens
[35]. About 2-fold higher values were found in primary open-angle glaucoma patients com-
pared to controls (cataract AH) [35], which is slightly more than the 1.4-fold higher value seen
in our PPCD patients compared to controls. Our results did not show any statistically signifi-
cant differences associated with the active TGF-β2 levels in the groups of PPCD_glaucoma
+ and PPCD_glaucoma- patients. It seems that the appearance of secondary glaucoma in our
PPCD patients was not directly associated with higher TGF-β2 levels in the AH. This indicates
that the higher levels of TGF-β2 in PPCD patients are not related to glaucoma itself, but to
changes occurring in PPCD. The reason for the higher levels of TGF-β2 (compared to control
levels) found in the AH of patients with both disorders (PPCD and open-angle glaucoma)
remains to be elucidated.
The mean levels of active TGF-β2 in our AH control samples obtained from cadaveric
donors (260.95 ± 112.43 pg/ml) are consistent with the mean levels of active TGF-β2 measured
in AH samples obtained from cataract surgery patients, which are frequently, but not exclu-
sively, used as control specimens [35, 40]. It has been reported that the freezing and thawing of
AH samples leads to a more than 4-fold increase of active TGF-β2 levels [27]. Although we
cannot exclude the influence of cryopreservation on the levels of TGF-β2 measured in our
samples, both control and pathological specimens were processed and stored in the same man-
ner, and therefore the statistical differences that we found should be actual and not an artifact
of cryopreservation. Due to the small volumes of the AH samples, we were not able to analyze
the levels of latent TGF-β2 and thus distinguish whether the higher levels of active TGF-β2 in
PPCD specimens are due to increased TGF-β2 activation or also due to increased TGF-β2
production.
In conclusion, our present study suggests that the higher levels of active TGF-β2 found in
the AH of PPCD patients compared to those in control samples could be a consequence of the
PPCD phenotype and can be considered as a feature characterizing this disease.
Supporting information
S1 File. Input data.
(XLSX)
Acknowledgments
The authors thank Viera Vesela, M.D., for her excellent technical assistance with the prepara-
tion of the specimens.
Author Contributions
Conceptualization: KJ LD.
Formal analysis: ZV.
The level of active TGF-β2 in AH of PPCD patients is higher compared to controls
PLOS ONE | https://doi.org/10.1371/journal.pone.0175509 April 17, 2017 8 / 11
Page 9
Funding acquisition: KJ.
Investigation: LD.
Methodology: KJ.
Project administration: KJ LD.
Resources: PS MF.
Supervision: KJ.
Validation: KJ.
Visualization: AS ZV.
Writing – original draft: AS KJ ZV.
Writing – review & editing: LD PS MF ZV KJ AS.
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